Self-assembling Peptide Research Articles

Evaluation of a Peptide Hydrogel as a Chondro-Instructive Three-Dimensional Microenvironment.

Articular cartilage injuries are inherently irreversible, even with the advancement in current therapeutic options. Alternative approaches, such as the use of mesenchymal stem/stromal cells (MSCs) and tissue engineering techniques, have gained prominence. MSCs represent an ideal source of cells due to their low immunogenicity, paracrine activity, and ability to differentiate. Among biomaterials, self-assembling peptide hydrogels (SAPH) are interesting given their characteristics such as good biocompatibility and tunable properties. Herein we associate human adipose-derived stem cells (hASCs) with a commercial SAPH, Puramatrix™, to evaluate how this three-dimensional microenvironment affects cell behavior and its ability to undergo chondrogenic differentiation. We demonstrate that the Puramatrix™ hydrogel comprises a highly porous matrix permissible for hASC adhesion and in vitro expansion. The morphology and cell growth dynamics of hASCs were affected when cultured on the hydrogel but had minimal alteration in their immunophenotype. Interestingly, hASCs spontaneously formed cell aggregates throughout culturing. Analysis of glycosaminoglycan production and gene expression revealed a noteworthy and donor-dependent trend suggesting that Puramatrix™ hydrogel may have a natural capacity to support the chondrogenic differentiation of hASCs. Altogether, the results provide a more comprehensive understanding of the potential applications and limitations of the Puramatrix™ hydrogel in developing functional cartilage tissue constructs.

Preserving Structurally Labile Peptide Nanosheets After Molecular Functionalization of the Self-Assembling Peptides.

A significant challenge in creating supramolecular materials is that conjugating molecular functionalities to building blocks often results in dissociation or undesired morphological transformation of their assemblies. Here we present a facile strategy to preserve structurally labile peptide assemblies after molecular modification of the self-assembling peptides. Sheet-forming peptides are designed to afford a staggered alignment with the segments bearing chemical modification sites protruding from the sheet surfaces. The staggered assembly allows for simultaneous separation of attached molecules from each other and from the underlying assembly motifs. Strikingly, using PEGs as the external molecules, PEG400 - and PEG700 -peptide conjugates directly self-associate into nanosheets with the PEG chains localized on the sheet surfaces. In contrast, the sheet formation based on in-register lateral packing of peptides does not recur upon the peptide PEGylation. This strategy allows for fabrication of densely modified assemblies with a variety of molecules, as demonstrated using biotin (hydrophobic molecule), c(RGDfK) (cyclic pentapeptide), and nucleic acid aptamer (negatively charged ssDNA). The staggered co-assembly also enables extended tunability of the amount/density of surface molecules, as exemplified by screening ligand-appended assemblies for cell targeting. This study paves the way for functionalization of historically challenging fragile assemblies while maintaining their overall morphology.

A General and Convenient Peptide Self-Assembling Mechanism for Developing Supramolecular Versatile Nanomaterials Based on The Biosynthetic Hybrid Amyloid-Resilin Protein.

Self-assembling peptides are valuable building blocks to fabricate supramolecular biomaterials, which have broad applications from biomedicine to biotechnology. However, limited choices to induce different globular proteins into hydrogels hinder these designs. Here, an easy-to-implement and tunable self-assembling strategy, which employs Ure2 amyloidogenic peptide, are described to induce any target proteins to assemble into supramolecular hydrogels alone or in combination with notable compositional control. Furthermore, the collective effect of nanoscale interactions among amyloid nanofibrils and partially disordered elastomeric polypeptides are investigated. This led to many useful macroscopic material properties simultaneously emerging from one pure protein material, i.e. strong adhesion to any substrates under wet conditions, rapidly self--assembling into robust and porous hydrogels, adaptation to remodeling processes, strongly promoting cell adhesion, proliferation and differentiation. Moreover, he demonstrated this supramolecular material's robust performance in vitro and vivo for tissue engineering, cosmetic and hemostasis applications and exhibited superior performance compared to corresponding commercial counterparts. To the best of his knowledge, few pure protein-based materials could meet such seemingly mutually exclusive properties simultaneously. Such versatility renders this novel supramolecular nanomaterial as next-generation functional protein-based materials, and he demonstrated the sequence level modulation of structural order and disorder as an untapped principle to design new proteins.

Potential Application of Self-Assembled Peptides and Proteins in Breast Cancer and Cervical Cancer

Ongoing research is gradually broadening the idea of cancer treatment, with attention being focused on nanoparticles to improve the stability, therapeutic efficacy, targeting, and other important metrics of conventional drugs and traditional drug delivery methods. Studies have demonstrated that drug delivery carriers based on biomaterials (e.g., protein nanoparticles and lipids) and inorganic materials (e.g., metal nanoparticles) have potential anticancer effects. Among these carriers, self-assembled proteins and peptides, which are highly biocompatible and easy to standardize and produce, are strong candidates for the preparation of anticancer drugs. Breast cancer (BC) and cervical cancer (CC) are two of the most common and deadly cancers in women. These cancers not only threaten lives globally but also put a heavy burden on the healthcare system. Despite advances in medical care, the incidence of these two cancers, particularly CC, which is almost entirely preventable, continues to rise, and the mortality rate remains steady. Therefore, there is still a need for in-depth research on these two cancers to develop more targeted, efficacious, and safe therapies. This paper reviews the types of self-assembling proteins and peptides (e.g., ferritin, albumin, and virus-like particles) and natural products (e.g., soy and paclitaxel) commonly used in the treatment of BC and CC and describes the types of drugs that can be delivered using self-assembling proteins and peptides as carriers (e.g., siRNAs, DNA, plasmids, and mRNAs). The mechanisms (including self-assembly) by which the natural products act on CC and BC are discussed. The mechanism of action of natural products on CC and BC and the mechanism of action of self-assembled proteins and peptides have many similarities (e.g., NF-KB and Wnt). Thus, natural products using self-assembled proteins and peptides as carriers show potential for the treatment of BC and CC.

Effects of Biodegradable Biomaterials Combined with Bone Marrow Mesenchymal Stem Cells Transplantation on Neurological Function in Rats with Ischemic Cerebral Infarction

The aim of this research was to explore the effects of degradable biomaterial self-polypeptide nanofiber scaffolds plus bone marrow mesenchymal stem cells (BMSCs) on the behavior and neurological function of rats with focal ischemic cerebral infarction (ICI). BMSCs were isolated and cultured. The self-assembling peptides and BMSCs were mixed to prepare self-assembling peptides/BMSCs (SAP/BMSCs) material. BMSCs and SAP/BMSCs were injected into the carotid artery of rats in Model group. The rats without ligation were used as Sham group. Longa 5 and modified neurological severity scores (mNSS) of each group were evaluated. The adhesive removal test and forelimb asymmetry test were used to examine the behavior of the rats. The brain tissues were collected, and the infarct size, neuronal apoptosis, and the protein expression of Nogo-A and lipoprotein a (LPA) in the hippocampus were detected by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining, TdT-mediated dUTP nick end labeling (TUNEL) staining, enzyme-linked immunosorbent assay (ELISA), and Western blot. The results indicated that the fiber diameter of the self-polymerizing peptide material was about 12 nm, and the length was about 100-250 nm. The cells grew well after composite BMSCs. Compared with Sham group, Longa 5, mNSS score, adhesive removal time, forelimb asymmetry test score, cerebral infarction volume, and neuronal apoptosis rate were increased, interleukin 6 (IL-6) and Tumor Necrosis Factor alpha (TNF-α) were raised, IL-10 was decreased, and Nogo-A and LPA protein was increased in Model group (P < 0.05). As against Model group, Longa 5, mNSS score, adhesive removal time, forelimb asymmetry test score, cerebral infarction volume, neuronal apoptosis rate, IL-6 and TNF-α were clearly decreased, and IL-10 was raised, and Nogo-A and LPA protein was clearly decreased in BMSCs and SAP/BMSCs groups (P < 0.05). As against BMSCs group, Longa 5, mNSS score, adhesive removal time, forelimb asymmetry test score, cerebral infarction volume, and neuronal apoptosis rate were clearly decreased, and IL-6 and TNF-α were decreased, and IL-10 was raised, and Nogo-A and LPA protein was decreased in SAP/BMSCs group (P < 0.05). Early transplantation of BMSCs can improve the neurobehavioral symptoms of ICI rats and inhibit neuronal apoptosis and inflammatory response. The effect of self-polymerizing peptide nanofiber scaffold plus BMSCs transplantation in ICI is superior to the transplantation of BMSCs alone.

Selectively Positioned Catechol Moiety Supports Ultrashort Self-Assembling Peptide Hydrogel Adhesion for Coral Restoration.

Coral reef survival is threatened globally. One way to restore this delicate ecosystem is to enhance coral growth by the controlled propagation of coral fragments. To be sustainable, this technique requires the use of biocompatible underwater adhesives. Hydrogels based on rationally designed ultrashort self-assembling peptides (USP) are of great interest for various biological and environmental applications, due to their biocompatibility and tunable mechanical properties. Implementing superior adhesion properties to the USP hydrogel compounds is crucial in both water and high ionic strength solutions and is relevant in medical and marine environmental applications such as coral regeneration. Some marine animals secrete large quantities of the aminoacids dopa and lysine to enhance their adhesion to wet surfaces. Therefore, the addition of catechol moieties to the USP sequence containing lysine (IIZK) should improve the adhesive properties of USP hydrogels. However, it is challenging to place the catechol moiety (Do) within the USP sequence at an optimal position without compromising the hydrogel self-assembly process and mechanical properties. Here, we demonstrate that, among three USP hydrogels, DoIIZK is the least adhesive and that the adhesiveness of the IIZDoK hydrogel is compromised by its poor mechanical properties. The best adhesion outcome was achieved using the IIZKDo hydrogel, the only one to show equally sound adhesive and mechanical properties. A mechanistic understanding of this outcome is presented here. This property was confirmed by the successful gluing of coral fragments by means of IIZKDo hydrogel that are still thriving after more than three years since the deployment. The validated biocompatibility of this underwater hydrogel glue suggests that it could be advantageously implemented for other applications, such as surgical interventions.

Self-Assembled Protein Nanostructures via Irreversible Peptide Assembly.

The quaternary structure of proteins extends the functionality of monomeric proteins. Similarly, self-assembled protein nanostructures (SPrNs) have great potential to improve the functionality and complexity of proteins; however, the difficulty associated with the fabrication of SPrNs is far greater than that associated with the fabrication of self-assembled peptides or polymers and often requires sophisticated computational design. To make the process of SPrN formation simpler and more intuitive, herein, we devise a strategy to adopt an irreversible self-assembled peptide nanostructure (SPeN) process en route to the formation of SPrNs. The strategy employs three sequential steps: first, the formation of SPeNs (an equilibrium process); second, covalent capture of SPeNs (an irreversible process); third, the final assembly of SPrNs via protein-peptide interactions (an equilibrium process). This strategy allowed us to fabricate SPrNs in which the size of the protein was approximately 9 times higher than that of the self-assembling peptide. Furthermore, we demonstrated that the irreversible SPeN could be used as a primary building block for assembly into superstructures. Overall, this strategy is conceptually as simple as SPeN fabrication and is potentially applicable to any soluble protein.

Calming the Nerves via the Immune Instructive Physiochemical Properties of Self-Assembling Peptide Hydrogels.

Current therapies for the devastating damage caused by traumatic brain injuries (TBI) are limited. This is in part due to poor drug efficacy to modulate neuroinflammation, angiogenesis and/or promoting neuroprotection and is the combined result of challenges in getting drugs across the blood brain barrier, in a targeted approach. The negative impact of the injured extracellular matrix (ECM) has been identified as a factor in restricting post-injury plasticity of residual neurons and is shown to reduce the functional integration of grafted cells. Therefore, new strategies are needed to manipulate the extracellular environment at the subacute phase to enhance brain regeneration. In this review, potential strategies are to be discussed for the treatment of TBI by using self-assembling peptide (SAP) hydrogels, fabricated via the rational design of supramolecular peptide scaffolds, as an artificial ECM which under the appropriate conditions yields a supramolecular hydrogel. Sequence selection of the peptides allows the tuning of these hydrogels' physical and biochemical properties such as charge, hydrophobicity, cell adhesiveness, stiffness, factor presentation, degradation profile and responsiveness to (external) stimuli. This review aims to facilitate the development of more intelligent biomaterials in the future to satisfy the parameters, requirements, and opportunities for the effective treatment of TBI.

Multiscale Simulations to Discover Self-Assembled Oligopeptides: A Benchmarking Study.

Peptide self-assembly is critical for biomedical and material discovery and production. While it is costly to experimentally test every possible peptide design, computational assessment provides an affordable solution to evaluate many designs and prioritize synthesis and characterization. Following a theoretical investigation, we present a systematic analysis of all-atom and coarse-grained simulations to predict peptide self-assembly. Benchmarking studies of two model dipeptides allow us to assess the impacts of intrinsic properties (such as amino acids and terminal modifications) and external environment (such as salinity) on the simulated aggregation. Further examination of 20 oligopeptides containing two to five amino acids shows good agreement among our theory, simulations, and prior experimental observations. The success rate of our prediction is 90%. Therefore, our theory, simulation, and analysis can be useful to identify peptide designs that can self-assemble and predict the potential nanostructures. These findings lay the ground for future virtual screening of peptide-assembled nanostructures and computer-aided biologics design.

Gel-to-Solution Transition of Sulfhydryl Self-Assembled Peptide Hydrogels Undergoing Oxidative Modulation.

The design of self-assembling biomaterials needs to take into consideration the timing and location of the self-assembly process. In recent decades, the principal strategy has been to control the peptide self-assembly under specific conditions to enable its functional performance. However, few studies have explored the responsive elimination of functional self-assembled peptide hydrogels after their function has been performed. We designed peptide ECAFF (ECF-5), which under reductive conditions can self-assemble into a hydrogel. Upon exposure to oxidizing conditions, disulfide bonds form between the peptides, altering their molecular structure and impacting their self-assembly capability. As a result, the peptide hydrogels transition to a soluble state. This study investigates the utilization of oxidation to induce a gel-to-solution transition in peptide hydrogels and provides an explanation for their degradation following free radical treatment. Self-assembled peptide hydrogel materials can be designed from a fresh perspective by considering the degradation that takes place after functional execution.

Development and evaluation of RADA-PDGF2 self-assembling peptide hydrogel for enhanced skin wound healing

Background: Wound healing complications affect numerous patients each year, creating significant economic and medical challenges. Currently, available methods are not fully effective in the treatment of chronic or complicated wounds; thus, new methods are constantly sought. Our previous studies showed that a peptide designated as PDGF2 derived from PDGF-BB could be a promising drug candidate for wound treatment and that RADA16-I can serve as a release system for bioactive peptides in wound healing. Based on that, in this work, we designed a new self-assembling hydrogel RADA-PDGF2, connecting both peptides by a sequence specific for neutrophil elastase, and evaluated its activity in wound healing.Methods: The physicochemical properties of the designed scaffold were analyzed using transmission electron microscopy, atomic force microscopy, cryoSEM microscopies, and circular dichroism spectroscopy. The enzymatic cleavage was performed using human neutrophil elastase and monitored using high-performance liquid chromatography and MS spectroscopic techniques. The aforementioned techniques (HPLC and MS) were also used to assess the stability of the peptide in water and human plasma. The biological activity was analyzed on human skin cells using a colorimetric XTT test, collagen synthesis evaluation, and a migration assay. The biocompatibility was analyzed with LDH cytotoxicity assay and flow cytometric analysis of activation of immune cells. Finally, RADA-PDGF2 activity in wound healing was checked in a mouse dorsal skin injury model.Results: The analysis showed that RADA-PDGF2 can self-assemble, form a hydrogel, and release a bioactive sequence when incubated with human elastase. It shows pro-proliferative and pro-migratory properties and accelerates wound closure in the mouse model compared to RADA16-I. In addition, it is not cytotoxic to human cells and does not show immunogenicity. RADA-PDGF2 seems to be a promising drug candidate for wound management.

Advances in Peptide-Decorated Targeted Drug Delivery: Exploring Therapeutic Potential and Nanocarrier Strategies.

Peptides are ideal biologicals for targeted drug delivery and have also been increasingly employed as theranostic tools in treating various diseases, including cancer, with minimal or no side effects. Owing to their receptor-specificity, peptide-mediated drug delivery aids in targeted drug delivery with better pharmacological biodistribution. Nanostructured self-assembled peptides and peptide-drug conjugates demonstrate enhanced stability and performance and captivating biological effects in comparison with conventional peptides. Moreover, they serve as valuable tools for establishing interfaces between drug carriers and biological systems, enabling the traversal of multiple biological barriers encountered by peptide-drug conjugates on their journeys to their intended targets. Peptide-based drugs play a pivotal role in the field of medicine and hold great promise for addressing a wide range of complex diseases such as cancer and autoimmune disorders. Nanotechnology has revolutionized the fields of medicine, biomedical engineering, biotechnology, and engineering sciences over the past two decades. With the help of nanotechnology, better delivery of peptides to the target site could be achieved by exploiting the small size, increased surface area, and passive targeting ability of the nanocarrier. Furthermore, nanocarriers also ensure safe delivery of the peptide moieties to the target site, protecting them from degradation. Nanobased peptide delivery systems would be of significant importance in the near future for the successful targeted and efficient delivery of peptides. This review focuses on peptide-drug conjugates and nanoparticle-mediated self-assembled peptide delivery systems in cancer therapeutics.

A repertoire of nanoengineered short peptide-based hydrogels and their applications in biotechnology

Peptide nanotechnology has currently bridged the gap between materials and biological worlds. Bioinspired self-assembly of short-peptide building blocks helps take the leap from molecules to materials by taking inspiration from nature. Owing to their intrinsic biocompatibility, high water content, and extracellular matrix mimicking fibrous morphology, hydrogels engineered from the self-assembly of short peptides exemplify the actualization of peptide nanotechnology into biomedical products. However, the weak mechanical property of these hydrogels jeopardizes their practical applications. Moreover, their functional diversity is limited since they comprise only one building block. Nanoengineering the networks of these hydrogels by incorporating small molecules, polymers, and inorganic/carbon nanomaterials can augment the mechanical properties while retaining their dynamic supramolecular nature. These additives interact with the peptide building blocks supramolecularly and may enhance the branching of the networks via coassembly or crystallographic mismatch. This phenomenon expands the functional diversity of these hydrogels by synergistically combining the attributes of the individual building blocks. This review highlights such nanoengineered peptide hydrogels and their applications in biotechnology. We have included exemplary works on supramolecular modification of the peptide hydrogel networks by integrating other small molecules, synthetic/biopolymers, conductive polymers, and inorganic/carbon nanomaterials and shed light on their various utilities focusing on biotechnology. We finally envision some future prospects in this highly active field of research.

Dosimetric characterization of double network Fricke hydrogel based on PVA-GTA and phenylalanine peptide derivative

A double network hydrogel based on Poly(vinyl-alcohol) (PVA) cross-linked with Glutaraldehyde (GTA) was recently developed by using self-assembling phenylalanine (Phe) peptide derivative (Fmoc-Phe-Phe-OMe), with the aim to improve the mechanical-elastic properties of PVA-GTA hydrogels. In this study, a characterization of the properties of Xylenol Orange based Fricke gel dosimeters obtained by infusing a Fricke solution into the double network hydrogel was performed. The gel dosimeters were irradiated with 6 MV and 15 MV X-rays produced by a medical linear accelerator and investigated by means optical absorbance measurements. The double network hydrogel formulation maintained a satisfactory level of radiological water-equivalence within the investigated radiotherapy range. Fricke gel dosimeters prepared with such network kept the desired properties of independence of the response of the dose rate and energy in the investigated intervals. Furthermore, the addition of self-assembling Phe peptide derivative proved not avoid the motion of radio-inducted ferric ions into the hydrogel, probably maintaining the main characteristics of the standard, no Phe peptide infused, formulation. The time course of formation of the optical response after the irradiation was observed to be similar to what previously measured in traditional PVA-GTA Fricke gel dosimeters, while a decrease of the sensitivity to radiation dose of the order of 30% was found. The extent of the decrease does not seem such as to impair the use of these dosimeters for evaluation of doses typical of radiation therapy applications. The overall dosimetric properties, coupled with the mechanical-elastic characteristics of the double network hydrogel, pave theway to the development of phantoms able both to mimic the deformation of organs possibly occurring during radiotherapy treatments and at the same time to assess the 3D dose distribution within such volumes.

Hypoxia-responsive calixarene-grafted self-assembled peptide hydrogel for inflammation suppression in ischemic stroke

Hypoxia is a significant characteristic of ischemic stroke. Recently, development of the pathology of tissue injury and deployment of new hypoxia-responsive technologies provide further opportunity for the application of hypoxia-responsive materials beyond cancer. To fully take advantage of hypoxic pathological feature of stroke and make advancement of stroke therapy, a hypoxia-responsive self-assembled peptide hydrogel was prepared for inflammation suppression in ischemic stroke treatment, utilizing azocalixarene (CA) as a hypoxia-responsive controlled release drug carrier. After induction of PBS, the peptide hydrogel was fabricated with brain-similar rheological modulus and shear-thinning property. The hypoxia-responsive performance of the peptide hydrogel was validated by three hypoxia & reductase systems including sodium hydrosulfite (SDT), rat liver microsomes and oxygen-glucose deprivation model (OGD) to achieve the responsive release of Cyanine 5-dimethyl (CY5-DM) or Fingolimod (FTY720) in vitro. After local administration of the hypoxia-responsive self-assembled peptide hydrogel, the improved motor function, reduced infarct volume and alleviated inflammation were achieved, revealing the effective stroke therapy in vivo.

Small Peptide-Based Nanodelivery Systems for Cancer Therapy and Diagnosis.

Developing nano-biomaterials with tunable topology, size, and surface characteristics has shown tremendously favorable benefits in various biologic and clinical applications. Among various nano-biomaterials, peptide-based drug delivery systems offer multiple merits over other synthetic systems due to their enhanced bio- and cytocompatibility and desirable biochemical and biophysical properties. Currently, around 100 peptide-based drugs are clinically available for numerous therapeutic purposes. In conjugation with chemotherapeutic moieties, peptides demonstrate a remarkable ability to reduce nonspecific drug effects by improving drug targetability at cancer sites. This review encompasses a wide-ranging role played by different peptide-based nanostructures in cancer theranostics. Section 1 introduces the rising concern about cancer as a disease and further describes peptide-based nanomaterials as biomedical agents to tackle the ailment. The subsequent section explores the mechanistic pathways behind the self-assembly of peptides to form hierarchically distinct assemblies. The crux of our review lies in an exhaustive exploration of the applications of various types of peptide-based nanostructures in cancer therapy and diagnosis. SIGNIFICANCE STATEMENT: Peptide-based drug delivery systems possess superior biocompatibility, biochemical, and biophysical properties compared to other synthetic alternatives. The development of these nano-biomaterials with customizable topology, size, and surface characteristics have shown promising outcomes in biomedical contexts. Peptides in conjunction with chemotherapeutic agents exhibit the ability to enhance drug targetability at cancer sites, reducing nonspecific drug effects. This comprehensive review emphasizes the pivotal role of diverse peptide-based nanostructures as cancer theranostics, elucidating their potential in revolutionizing cancer therapy and diagnosis.

A methodological approach by capillary electrophoresis coupled to mass spectrometry via electrospray interface for the characterization of short synthetic peptides towards the conception of self-assembled nanotheranostic agents

Nanostructures formed by the self-assembling peptide building blocks are attractive materials for the design of theranostic objects due to their intrinsic biocompatibility, accessible surface chemistry as well as cavitary morphology. Short peptide synthesis and modification are straightforward and give access to a great diversity of sequences, making them very versatile building blocks allowing for the design of thoroughly controlled self-assembled nanostructures. In this work, we developed a new CE-DAD-ESI-MS method to characterize short synthetic amphiphilic peptides in terms of exact sequence and purity level in the low 0.1 mg.mL−1 range, without sample treatment. This study was conducted using a model sequence, described to have pH sensitive self-assembling property. Peptide samples obtained from different synthesis processes (batch or flow, purified or not) were thus separated by capillary zone electrophoresis (CZE). The associated dual UV and MS detection mode allowed to evidence the exact sequence together with the presence of impurities, identified as truncated or non-deprotected sequences, and to quantify their relative proportion in the peptide mixture. Our results demonstrate that the developed CE-DAD-ESI-MS method could be directly applied to the characterization of crude synthetic peptide products, in parallel with the optimization of peptide synthetic pathway to obtain controlled sequences with high synthetic yield and purity, which is crucial for further design of robust peptide based self-assembled nanoarchitectures.

Adventitial delivery of miR-145 to treat intimal hyperplasia post vascular injuries through injectable and in-situ self-assembling peptide hydrogels

Intimal hyperplasia is a common lesion that can be observed in diverse vascular diseases. Drug-eluting stents and drug-coated balloons, which can release anti-proliferative agents to inhibit smooth muscle cell (SMC) proliferation, are developed to prevent intimal hyperplasia. However, these intervention devices still cannot achieve satisfactory clinical outcomes. In contrast to endovascular drug delivery, vascular adventitial drug delivery is a new strategy. To develop a vascular adventitial drug delivery system to treat intimal hyperplasia post vascular injuries, we loaded miR-145-5p-agomir (miR-145) into an injectable and in-situ self-assembling RAD peptide hydrogel. In vitro data showed that the miR-145 could be well incorporated into the RAD peptide hydrogels and released in a slow and controlled manner. The released miR-145 could transfect SMCs successfully, and the transfected SMCs exhibited a reduced migration capacity and higher expressions of SMC contractile biomarkers as compared to the non-transfected SMCs. In vivo data showed that the retention of the miR-145 was greatly elongated by the RAD peptide hydrogels. In addition, the application of the miR-145-loaded RAD peptide hydrogels surrounding injured arteries decreased the proliferative SMCs, promoted the regeneration of endothelium, reduced the macrophage infiltration, inhibited the neointimal formation and prevented adverse ECM remodeling via downregulation of KLF4 expression. The RAD peptide hydrogels loaded with miR-145 can successfully inhibit intimal hyperplasia after vascular injuries and thus hold great potential as an innovative extravascular drug delivery approach to treat vascular diseases. Statement of significanceIntimal hyperplasia is a common lesion that can be observed in diverse vascular diseases. Drug-eluting stents and drug-coated balloons, which can release anti-proliferative agents to inhibit smooth muscle cell (SMC) proliferation, are developed to prevent intimal hyperplasia. However, these intervention devices still cannot achieve satisfactory clinical outcomes. In contrast to endovascular drug delivery, vascular adventitial drug delivery is a new strategy. Our work here demonstrates that the RAD peptide hydrogels loaded with miR-145-5p-agomir (miR-145) can successfully reverse intimal hyperplasia after vascular injuries and thus hold great potential as an innovative vascular adventitial drug delivery approach to treat vascular diseases. Our work proposes a possible paradigm shift from endovascular drug delivery to extravascular drug delivery for vascular disorder treatment.

A novel protein purification scheme based on salt inducible self-assembling peptides

BackgroundProtein purification remains a critical need for biosciences and biotechnology. It frequently requires multiple rounds of chromatographic steps that are expensive and time-consuming. Our lab previously reported a cleavable self-aggregating tag (cSAT) scheme for streamlined protein expression and purification. The tag consists of a self-assembling peptide (SAP) and a controllable self-cleaving intein. The SAP drives the target protein into an active aggregate, then by intein-mediated cleavage, the target protein is released. Here we report a novel cSAT scheme in which the self-assembling peptide is replaced with a salt inducible self-assembling peptide. This allows a target protein to be expressed first in the soluble form, and the addition of salt then drives the target protein into the aggregated form, followed by cleavage and release.ResultsIn this study, we used MpA (MKQLEDKIEELLSKAAMKQLEDKIEELLSK) as a second class of self-assembling peptide in the cSAT scheme. This scheme utilizes low salt concentration to keep the fusion protein soluble, while eliminating insoluble cellular matters by centrifugation. Salt then triggers MpA-mediated self-aggregation of the fusion, removing soluble background host cell proteins. Finally, intein-mediated cleavage releases the target protein into solution. As a proof-of-concept, we successfully purified four proteins and peptides (human growth hormone, 22.1 kDa; LCB3, 7.7 kDa; SpyCatcherΔN-ELP-SpyCatcherΔN, 26.2 kDa; and xylanase, 45.3 kDa) with yields ranging from 12 to 87 mg/L. This was comparable to the classical His-tag method both in yield and purity (72–97%), but without the His-tag. By using a further two-step column purification process that included ion-exchange chromatography and size-exclusion chromatography, the purity was increased to over 99%.ConclusionOur results demonstrate that a salt-inducible self-assembling peptide can serve as a controllable aggregating tag, which might be advantageous in applications where soluble expression of the target protein is preferred. This work also demonstrates the potential and advantages of utilizing salt inducible self-assembling peptides for protein separation.

Insights into diphenylalanine peptide self-assembled nanostructures for integration as nanoplatforms in analytical and medical devices

New insights on the self-assembling process of diphenylalanine (FF) into nanostructures in view of its application as an alternative nanomaterial for bioanalytical and biomedical systems are presented in the frame of the present work. Experimental conditions, such as peptide concentration and solubilization medium pH, were explored to understand the hierarchical process involved in the formation of self-assembled nanostructures arising from the simple and short diphenylalanine peptide. Optical microscopy and TEM images supported by DLS data authenticated the hierarchical self-assembly outcoming from the original nature of the first nanostructures, showing individual nanotubes and vesicles stacking to grow well-defined microtubes. Moreover, the influence of metal cations on peptide self-assembly was evaluated for the first time in the presence of Mg2+ and compared with other ions, such as Na+, K+, and Ca2+. The results evidenced a tendency of Mg2+ to interact with diphenylalanine peptides to form self-assembled nanostructures showing vesicle- and ellipse-like morphologies. FF solubilization in water prepared under sonication in a bath at 65–68 °C followed by dilution into chloride metal cation solutions at 50 mmol·L−1 proved to be optimal conditions to obtain metal-coordinated self-assembled FF structures. Besides, the latter revealed fluorescence features and electron-transfer properties on carbon-based electrode surfaces, that can be further explored in analytical and bioanalytical devices for fully integrated platforms. In this context, self-assembled nanostructures achieved in the presence of Mg2+ and Ca2+ were implemented for the surface modification of carbon screen-printed electrodes and proved to increase the electrochemical response toward a redox probe. This proof of concept is particularly interesting for further use of these peptide-based nanoarchitectures as nanoplatforms for clinical imaging, therapeutic and diagnosis purposes.